Chymosin, also known as rennin, is a commercially important enzymatic protein, commonly used in the cheese manufacturing industry to coagulate milk. Traditionally chymosin has been prepared from its natural source, the fourth stomach of unweaned calves, although recovery from the stomachs of other mammals, such as lamb, goats etc. heretofore was known. More recently, primarily as a result of a decrease in calf production, recombinant DNA techniques have been employed to produce chymosin by fermentation in genetically engineered microorganisms. Thus a variety of bacterial and fungal hosts have been genetically modified to produce chymosin by fermentation, including for example, the bacterial hosts Escherichia coli, (European Patent 0 134 662 A1; Nishimori et al. (1982) J. Biochem 91: 1085-1088), Bacillus subtilis (U.S. Pat. No. 5,624,819; U.S. Pat. No. 5,716,807 and Parente et al. (1991) FEMS 77: 243-250) and the fungal hosts Aspergillus sp. (European Patent 0 575 462 B1; U.S. Pat. Nos. 5,364,770 and 5,863,759; Cullen et al. (1987) Bio/Technology 5: 369-375, Dunn-Coleman et al. (1991) Bio/Technology 9: 976-981., and Tsuchiua et al. (1993) Appl. Microbial Biotech. 40: 327-332), Kluyveromyces lactis (van der Berg et al. (1990) Bio/Technology 8: 135-139 and Trichoderma ressei (Jarkki et al. (1989) Bio/Technology 7: 596-603; Pitts et al. (1991) Biochemical Society Transactions 19: 663-665). As well, more general expression in fungi, yeast and bacteria (U.S. Pat. No. 4,666,847) and in filamentous fungi (U.S. Pat. No. 5,578,463).
The active enzyme chymosin (E.C. 3.4.23.4) is comprised of a polypeptide chain of a molecular mass of 35.6 kDa. However crude extracts of calf stomach mucosa in addition to active chymosin, contain two inactive precursor polypeptides known as pre-pro-chymosin and pro-chymosin. Pre-pro-chymosin contains an extra 58 amino acids at the N-terminus, whereas pro-chymosin contains an extra 42 amino acids. Conversion of the inactive precursor protein into enzymatically active chymosin requires the step-wise removal of the chymosin pre-peptide and pro-peptide. In vivo these activation steps take place in the calf stomach. The chymosin pre-peptide directs secretion of the polypeptide by the stomach cells and is removed upon secretion of the polypeptide by the stomach cells. The chymosin pro-peptide is subsequently removed in the gastric lumen, thereby activating the enzyme. The activation reaction can also be performed in vitro at pH values below 5. With regards to the enzyme chymosin, it should further be noted that chymosin purified from calf stomach is a mixture of two different polypeptides known as chymosin A and chymosin B. Both of these polypeptides are active and differ only with respect to one amino acid. The amino acid residue at position #290 is an aspartate residue in chymosin A and a glycine residue in chymosin B (Foltman et al., (1977) Proc. Natl. Acad. Sci. USA 74: 2331-2324; Foltman et al., (1979) J. Biol. Chem. 254: 8447-8456).
There are several disadvantages associated with the recombinant production of chymosin in fermentation systems. In general, fermentation systems require the use of large fermentation vessels that have both large space and energy requirements and consequently are costly. As well, the growth media require large volumes of water and may require special chemicals. Both of these may present environmental issues in the disposal of the large amounts of potentially harmful waste. Further, storage and shipment of raw material containing chymosin is problematic. The bacterial or fungal fermentation broth need to be processed immediately or refrigerated in large volumes since the enzyme is not stable for long periods in the broth.
The use of plants as bioreactors for the commercial production of recombinant proteins is well known. For example, avidin, β-glucuronidase and aprotinin (see patents U.S. Pat. Nos. 5,767,379, 5,804,694 and 5,824,870) have been recombinantly expressed in corn. Further, U.S. Pat. Nos. 5,543,576 and 5,714,474 are broadly directed to the recombinant production of enzymes in seeds and to the use of seeds or milled seeds comprising enzymes as a raw material in the preparation of food and feed products. Although U.S. Pat. Nos. 5,543,576 and 5,714,474 suggest chymosin as one potential enzyme that may be produced in seeds, there is no reduction to practice. These patents are further limited by the fact that in order to use the chymosin for the commercial production of cheese, chymosin would have to be purified from the seed or milled seeds.
PCT patent application WO 92/01042 discloses the expression of chymosin in the leaves of transgenic tobacco and potato plants. According to the disclosure chymosin expression levels of only 0.1% to 0.5% (w/w) of total soluble leaf protein were attained. The methodology of WO 92/01042 is further limited in that the production in leaves would require immediate extraction of the enzyme from the leaf material upon harvesting of the plants as the enzyme would lose activity when stored in leaves. In addition, due to the relatively high water content of leaves, large amounts of biomass must be processed.
There is a need in the art to further improve methods for the recombinant expression of chymosin in plants.